The innate immune system is the first line of defense against infection and is thought to primarily be mediated by phagocytic immune cells such as macrophages and dendritic cells. These cells recognize microorganisms via a limited number of germline-encoded pattern recognition receptors (PRRs) that recognize microbial components known as pathogen-associated molecular patterns, which are essential for the survival of the microorganism and, therefore, difficult for the microorganism to alter.
Several classes of PRRs, including cell surface-located Toll-like receptors (TLRs) and cytoplasmic receptors, recognize distinct microbial components and directly activate immune cells, triggering intracellular signaling cascades that rapidly induce the expression of a variety of inflammatory cytokines that initiate a variety of overlapping immune responses. One of the best known PRRs is TLR4, which recognizes the major Gram-negative bacterial surface component lipopolysaccharide (LPS) [Akira et al., Cell 124:783-801 (2006)]. See also, Beutler, Blood, 113:1399-1407 (2009), and Moresco et al., Curr. Biol. 21(13):R488-93, (2011) and the citations therein for a historical perspective of the research done in finding TLRs and determining the function of TLRs.
Most of the TLRs are functional multimers. Some are heteromeric. Some appear to be homomeric, and in some cases, non-TLR subunits are part of the signaling complex. For example, TLR4 seems not to detect LPS directly, but only as a complex with MD-2, a small secreted protein that is tightly associated with the TLR4 ectodomain. Crystallographic analysis has shown the nature of the interaction between specific TLR ligands and the Toll-like receptors, including interactions between LPS and the MD-2:TLR4 complex. Beutler, Blood 113:1399-1407 (2009).
Studies on TLR4 signaling in monocytes, macrophages, and dendritic cells have revealed that engagement of the MD-2:TLR4 complex (hereinafter just “TLR4”, for ease in expression) by LPS triggers a signaling cascade involving several intracytoplasmic and nuclear transcriptional factors. TLR4 activation first engages a set of adaptor family members that link TLR4 to the serine/threonine kinases. These kinases mediate phosphorylation and ubiquitination of various substrates, eventually resulting in the activation of the transcriptional factor NF-κB, which regulates the expression of several immunomodulatory cytokines [Kawai et al., Cell Death Differ 13:816-825 (2006)].
Freund's adjuvant (mycobacteria in mineral or vegetable oils), aluminum hydroxide (“alum”), and LPS (lipopolysaccharide) have been used to augment antibody responses to co-administered proteins. In the US, only alum is approved for use in human vaccines.
Of these adjuvants, only LPS and its derived lipid A have a well-defined target—TLR4, but the toxicity of these TLR4 ligands is considerable as is their instability in vivo and they are not easily conjugated to antigens.
In work that provides a powerful paradigm for synthetic “unnatural” adjuvant discovery, we identified a new class of robust small molecule adjuvants that: (1) emerged from screening an α-helix mimetic library, (2) act by a well-defined mechanism (TLR4 agonist), (3) are easy to produce and structurally manipulate, (4) are non-toxic, and (5) elicit improved and qualitatively different responses from LPS even though they share the same receptor. Such adjuvants may be used for co-administration or for covalently-tethered vaccination against any microbe susceptible to antibody-based protective immunization.